D-alanylation of lipoteichoic acid: role of the D-alanyl carrier protein in acylation

Abstract

The D-alanylation of membrane-associated lipoteichoic acid (LTA) in gram-positive organisms requires the D-alanine-D-alanyl carrier protein ligase (AMP) (Dcl) and the D-alanyl carrier protein (Dcp). The dlt operon encoding these proteins (dltA and dltC) also includes dltB and dltD. dltB encodes a putative transport system, while dltD encodes a protein which facilitates the binding of Dcp and Dcl for ligation with D-alanine and has thioesterase activity for mischarged D-alanyl-acyl carrier proteins (ACPs). In previous results it was shown that D-alanyl-Dcp donates its ester residue to membrane-associated LTA (M. P. Heaton and F. C. Neuhaus, J. Bacteriol. 176: 681-690, 1994). However, all efforts to identify an enzyme which catalyzes this D-alanylation process were unsuccessful. It was discovered that incubation of D-alanyl-Dcp in the presence of LTA resulted in the time-dependent hydrolysis of this D-alanyl thioester. D-Alanyl-ACP in the presence of LTA was not hydrolyzed. When Dcp was incubated with membrane-associated D-alanyl LTA, a time and concentration-dependent formation of D-alanyl-Dcp was found. The addition of NaCl to this reaction inhibited the formation of D-alanyl-Dcp and stimulated the hydrolysis of D-alanyl-Dcp. Since these reactions are specific for the carrier protein (Dcp), it is suggested that Dcp has a unique binding site which interacts with the poly(Gro-P) moiety of LTA. It is this specific interaction that provides the functional specificity for the D-alanylation process. The reversibility of this process provides a mechanism for the transacylation of the D-alanyl ester residues between LTA and wall teichoic acid.

FIG. 1

Hydrolysis of d-[¹⁴C]alanyl-Dcp in the presence of LTA. The reaction mixture contained 4.4 μM LTA-phosphorus from S. aureus, 8 μM d-[¹⁴C]alanyl-Dcp (6,700 cpm/nmol), and 30 mM bis-Tris (pH 6.5) in a total of 350 μl. Samples (50 μl) were removed and added to 950 μl of 10% TCA. The amount of radiolabeled d-alanyl-Dcp was determined as described in Materials and Methods, and the amount of d-alanine released was determined by subtraction.

FIG. 2

Effect of increasing d-alanyl-Dcp on the velocity of hydrolytic cleavage. The reaction mixture contained increasing concentrations of d-[¹⁴C]alanyl-Dcp in the presence of 4.4 μM LTA-phosphorus in the reaction mixture described in the legend to Fig. 1. The mixtures were incubated for 5 min prior to termination with 10% TCA, and the amounts of d-alanyl-Dcp remaining were determined for calculating the initial velocities of d-alanine released. In the inset a double-reciprocal plot is presented from which V_max (5.0 μM/min) and K_m (7.5 × 10⁻⁶ M) were calculated.

FIG. 3

Effect of increasing LTA-phosphorus on the hydrolysis of d-[¹⁴C]alanyl-Dcp. The reaction mixtures (50 μl) contained the indicated concentrations of LTA-phosphorus from S. aureus, 8 μM d-[¹⁴C]alanyl-Dcp (6,700 cpm/nmol), and 30 mM bis-Tris (pH 6.5). The amounts of d-alanine released were determined as described in the legend to Fig. 1.

FIG. 4

Inhibition of d-alanyl-Dcp hydrolysis. The reaction mixture contained 4.4 μM LTA-phosphorus and 8 μM d-[¹⁴C]alanyl-Dcp (6,700 cpm/nmol) in the presence of either 85 μM apo-Dcp or 85 μM apo-ACP. Samples were removed at the indicated times, and the amounts of d-alanyl-Dcp or d-alanyl-ACP were determined as described in Materials and Methods. The amounts of d-alanine released were determined as described in the legend to Fig. 1.

FIG. 5

(A) Effect of pH on the hydrolysis of d-alanyl-Dcp in the presence of LTA. (B) Effect of temperature on the specific and nonspecific hydrolyses of d-alanyl-Dcp and d-alanyl-ACP in the presence of LTA. The reaction mixtures for panel A contained either 30 mM sodium acetate (pH 3.0 to 5.0), bis-Tris (pH 6.0 to 7.0), or Tris-HCl (pH 7.5 to 9.5) in the presence of 4.4 μM LTA-phosphorus and 8 μM d-[¹⁴C]alanyl-Dcp. The reaction mixtures for panel B contained 30 mM bis-Tris (pH 6.5), either 8 μM d-[¹⁴C]alanyl-Dcp or d-[¹⁴C]alanyl-ACP, and 4.4 μM LTA-phosphorus where indicated. The reaction mixtures for panel A were incubated for 5 min at 37°C and at the indicated temperature for panel B. The amounts of d-alanyl-Dcp remaining were determined as described in Materials and Methods. In panel A, 100% activity is given by the velocity at pH 6.5 in the presence of LTA. In panel B, 100% activity is given by the amount of d-[¹⁴C]alanyl-Dcp added to the reaction mixture. The amount of specific hydrolysis is the difference between the hydrolyses observed for d-alanyl-Dcp (LTA) and d-alanyl-Dcp (no LTA).

FIG. 6

Effect of Dcp concentration on the formation of d-alanyl-Dcp from membrane-associated d-alanyl-LTA. The reaction mixture contained 20 μg of membrane-associated d-[¹⁴C]alanyl-LTA (6,700 cpm/nmol), the indicated concentration of Dcp or ACP, and 30 mM bis-Tris buffer (pH 6.5) in a total volume of 15 μl. It was incubated for 30 min at 37°C. The amounts of d-[¹⁴C]alanyl-Dcp formed were quantified by nondenaturing polyacrylamide gel electrophoresis by the method of Heaton and Neuhaus (12).

FIG. 7

Hydrolysis of d-[¹⁴C]alanyl-Dcp by membranes in the presence of increasing concentrations of NaCl. Because d-alanyl-Dcp can transfer its activated d-alanine to membrane-associated LTA, the ordinate represents the aggregate of d-alanyl-Dcp and d-alanyl-LTA. The reaction mixtures contained 30 mM bis-Tris (pH 6.5) and 0.25 mg of membranes from either L. casei 102S or the L. casei 102S dltD::cat mutant (5) per ml in the presence of increasing concentrations of NaCl in a volume of 50 μl. The reaction time was 30 min at 37°C. The amount of hydrolysis was quantified by the procedure described in Materials and Methods.

FIG. 8

Effect of increasing concentrations of NaCl on the formation of d-[¹⁴C]alanyl-Dcp from membrane-associated d-[¹⁴C]alanyl-LTA and Dcp. The reaction mixture contained 30 mM bis-Tris (pH 6.5) and 20 μg of d-[¹⁴C]alanyl-LTA (6,700 cpm/nmol) in a volume of 15 μl. The reaction time was 20 min at 37°C. The reaction mixtures were desalted using Nanosep microconcentrators. In the reaction mixture designated “Dialyzed” the d-[¹⁴C]alanyl-LTA was treated with 1.0 M NaCl for 20 min and then dialyzed before incubation with Dcp by using the Nanosep concentrator (10K). It was incubated in the reaction mixture described above for the indicated time. The amount of d-alanyl-Dcp formed was quantified by nondenaturing polyacrylamide gel electrophoresis (12).

FIG. 9

Proposed mechanisms for the hydrolysis of d-alanyl-Dcp in the presence of purified LTA (A), the formation of d-alanyl-Dcp from membrane-associated d-alanyl-LTA (B), and the formation of membrane-associated d-alanyl-LTA from d-alanyl-Dcp (C). B·· and ∶B indicate an unknown proton acceptor for generating the nucleophile. The electrostatic interaction between Dcp phosphodiester anion may be the result of Arg-64 (see Discussion). Events up to the tetrahedral intermediate stage are shown.